Use of Super-Resolution Optical Microscopy To Reveal Direct Virus Binding at Hybrid Core–Shell Matrixes

Abstract

Polymer particles with antibody-like affinity, i.e., molecularly imprinted polymers, offer an ideal platform for biopharmaceutical virus purification. In recent years, attempts combining molecular imprinting technology with a variety of visualization and detection techniques have been reported for directly confirming the localized presence of the template. Direct target visualization is crucial for the characterization of molecularly imprinted polymers, especially if biological templates such as viruses are used. In the present study, for the first time the viral binding behavior at virus-imprinted polymers (VIPs) via stimulated emission depletion (STED) microscopy is shown by imaging individual, fluorescently labeled virus particles. STED microscopy achieves among various other super-resolution techniques the best temporal resolution at high spatial resolution. An innovative virus purification material selective for human adenovirus type 5 (AdV5) offered highly purified virus for the subsequent fluorescent labeling procedure, thus enabling STED imaging. Excellent binding affinities (150-fold higher versus control particles) and high selectivity toward the target virus (AdV5) were observed at those VIPs, even in competitive binding experiments with minute virus of mice using dual-label STED microscopy.

Towards a Quantitative Single Particle Characterization by Super Resolution Microscopy: From Virus Structures to Antivirals Design

“…Lastly, molecularly imprinted polymers (MIPs) have been studied for detection and purification of viruses that could bind with high affinity and specificity. Using STED, Gast et al (2020) characterized the binding affinity and stability of adenovirus on the detection beads MIP, concluding that 30-40 adenovirus particles could bind in a mechanically stable way, proving that this detection system is robust, versatile, stable, and cost-efficient (Gast et al, 2020).…”

Virus morphology: Insights from super-resolution fluorescence microscopy

“…Smaller tags such as nanobodies or click chemistry with organic fluorophores may help to resolve this issue. More recent alternatives include single-molecule fluorescence in situ hybridization (smFISH) which uses fluorescently labelled DNA or RNA probes to hybridise the viral genome [E.g., [ 20 ]], molecularly imprinted polymers (MIPs) [ 21 ] or a near-instantaneous cation-mediated labelling method that binds small, non-specific fluorescent DNAS to the surface of enveloped viruses [ 22 , 23 ].…”

Advanced Nanomaterials for Preparedness Against (Re‐)Emerging Viral Diseases

“…These nanomaterials, paired with super-resolution optical imaging techniques, further suggest a useful tool for understanding the fundamentals of virus structures and mechanisms of action. [17] The biochemical systems of viruses, where they adapt the ability to enter cells, disassemble, and deliver their genomes and proteins, have been adopted to develop efficient vaccines or antiviral agents. All these unique nanoscale features and extraordinary surface properties have made nanomaterial-based treatment and diagnostic strategy of several pathologies highly effective.…”